Ribonucleases such as ribonuclease A ("RNase A") and their cytotoxicity toward tumor cells are well documented from studies performed in the 1960s and 1970s and reviewed in Roth, J., 1963, Cancer Res. 23:657-666. Human serum was also discovered to contain several RNases (Reddi, E., 1975, Biochem. Biophys. Res. Commun. 67:110-118, Blank et al., Human body fluid ribonucleases: detection, interrelationships and significance 1-203-209 (IRL Press, London, 1981)) that are expressed in a tissue specific manner. The proteins involved in the host defense activity of the eosinophil are homologous to RNases and express RNase activity (Gleich et al., 1986, Proc. Natl. Acad. Sci., USA 83:3146-3150; Slifman et al., 1986, J. Immunol, 137:2913-2917). Thus, human serum RNases were believed to also have host defense activities.
Further to these early studies was the discovery that an anti-tumor protein from oocytes of Rana pipiens has homology to RNase A (Ardelt et al., 1991, J. Biol. Chem. 266:245-251). This protein has been termed ONCONASE.RTM., Alfacell Corporation, N.J. See also e.g., Darzynkiewicz et al. (1988) Cell Tissue Kinet. 21, 169-182, Mikulski et al. (1990) Cell Tissue Kinet. 23, 237-246. This protein is also described in U.S. Pat. No. 4,888,172. Phase I and Phase I/II clinical trials of ONCONASE.RTM. as a single therapeutic agent in patients with a variety of solid tumors (Mikulski et al. (1993) Int. J. of Oncology 3, 57-64) or combined with tamoxifen in patients with advanced pancreatic carcinoma have recently been completed (Chun et al. (1995) Proc Amer Soc Clin Oncol 14 No. 157, 210). Conjugation of ONCONASE.RTM. to cell-type-specific ligands increased its potency towards tumor cells (Rybak et al. (1993) Drug Delivery 1, 3-10). Taken together, these results indicate that ONCONASE.RTM. has properties that are advantageous for the generation of a potent selective cell killing agent.
However, since this is not a human-derived protein, it is prone to stimulating undesirable immune responses when used in humans. Thus, it would be desirable to retain the potent cytotoxic properties of this molecule while reducing its immunogenicity in humans. Further, it would be desirable to produce derivations of this molecule recombinantly so that it may be better chemically conjugated or recombinantly joined to other molecules for targeting to specific cells. Until the invention described herein, it has proven difficult to recombinantly express an active cytotoxic molecule related to ONCONASE.RTM.. Though it was thought that the methionine-glutamic acid amino terminal end of the recombinant molecule prohibited the molecule from having significant enzymatic activity, a means to solve this problem has not been forthcoming until the invention herein.
Further, although advances in protein design techniques promise to alleviate some of the immunogenicity associated with the antibody portion of immunotoxins (Bird et al., 1988, Science 242:423; Huston et al., 1988, Proc Natl Acad Sci USA 85:5879; Ward et al., 1989, Nature 341:544), no solution has been forthcoming for the immunogenicity of the toxin portion other than immunosuppression of the patients (Khazaeli et al., 1988, Proceedings of AACR 29:418). Thus, there is a continuing need for methods and compositions that would reduce the immunogenicity of the Rana pipiens-derived toxic moiety.
Non-cytotoxic human members of the RNase A superfamily linked to tumor associated antigens by chemical (Rybak et al.(1991) J. Biol. Chem 266, 1202-21207, Newton et al. (1992) J. Biol. Chem. 267, 19572-19578) or recombinant means (Rybak et al. Proc. Natl. Acad. Sci. U.S.A. 89, 3165, Newton et al. (1994) J Biol Chem. 269, 26739-26745 have been shown to offer a strategy for selectively killing tumor cells with less immunogenicity than current strategies employing plant and bacterial toxins Rybak, S. M. & Youle, R. J. (1991) Immunol. and Allergy Clinics of North America 11:2, 359-380. Human-derived ribonucleases of interest include eosinophil-derived neurotoxin (EDN) and angiogenin.